Methods and compositions for treating conditions related to lack of blood supply, shock, and neuronal injuries

ABSTRACT

A pharmaceutical composition comprising a lipid component; an amphiphilic emulsifier; and a polar liquid carrier. The lipid component and the amphiphilic emulsifier form free-moving lipid-carrying micelles (LMs) in the polar liquid carrier. The pharmaceutical composition is free of hemoglobin and fluorocarbon and can be used for treating conditions related to lack of blood supply and to raise the blood pressure.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/273,471, filed on Oct. 14, 2011, now allowed, which is acontinuation-in-part of U.S. application Ser. No. 12/314,737, filed onDec. 16, 2008, now U.S. Pat. No. 8,063,020, which claims the priority ofU.S. Provisional Application No. 61/016,443, filed on Dec. 22, 2007 andU.S. Provisional Application No. 61/064,639, filed on Mar. 18, 2008;U.S. application Ser. No. 13/273,471 is also a continuation-in-part ofU.S. application Ser. No. 12/696,107, filed on Jan. 29, 2010, whichclaims priority of U.S. Provisional Application Ser. No. 61/202,124filed Jan. 30, 2009; U.S. application Ser. No. 13/273,471 also claimspriority of U.S. Provisional Application No. 61/432,919, filed on Jan.14, 2011 and U.S. Provisional Application No. 61/490,816, filed on May27, 2011. All of the aforementioned applications are incorporated hereinby reference in their entirety.

FIELD

The technical field is medical treatment and, in particular, methods andcompositions for treating conditions related to lack of blood supply,shock and neuronal injuries.

BACKGROUND

Shock is a consequence of inadequate circulation due to causes thatinclude blood loss, vasodilation or dehydration. The loss of circulationdeprives cells of oxygen and nutrients and activates a powerfulinflammatory response. Such states occur after injury due to war,accident, and assault resulting in massive blood loss or due to massiveinfection. The current therapeutic approach is to rapidly infuse fluidsin order to restore circulating volume. This rapid infusion of fluidsmay be combined with pharmacological agents that directly constrictblood vessels. Both the infusion of fluids and the administration ofdrugs that constrict blood vessels cause an increase in the bloodpressure. However, the pathophysiology of shock is far more complex thanthat which can be remedied by simply adding volume and clamping down theblood vessels to create a higher blood pressure. Once shock has occurredand the longer it persists numerous toxic substances termed mediatorsare released into the blood stream. These mediators create a dangerousstate that is markedly different from that which existed prior to theevent that led to shock.

Current methods for treating shock are of limited efficacy. Examples ofresuscitation fluids are either crystalloids that contain various saltssuch as Ringer's lactate that rapidly diffuses out of the bloodstreamand those that contain colloids or large molecules that remain in thebloodstream a longer time such as those that contain Hetastarch, aplasma volume expander derived from natural sources of starch. Numerouscomplications have been observed with both. Hetastarch enhancesbleeding, a counterproductive property after massive blood loss.Overreliance on vasoconstrictors will raise the blood pressure butdecrease tissue perfusion leading to tissue necrosis and death.

Therefore, there exists a need for a low-cost therapeutic volumeexpander that is capable of bringing oxygen and nutrients to the tissueand reduce inflammatory responses.

SUMMARY

One aspect of the present invention relates to a pharmaceuticalcomposition comprising a lipid component, an amphiphilic emulsifier, anda polar liquid carrier. The lipid component and the amphiphilicemulsifier form free-moving lipid-carrying micelles (LMs) in the polarliquid carrier. The pharmaceutical composition is free of hemoglobin andfluorocarbon.

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising 10%-80% (w/v) of a lipid component selected fromthe group consisting of soybean oil, chia bean oil and algae oil; 1%-5%(w/v) of an amphiphilic emulsifier selected from the group consisting ofegg yolk phospholipids and α-phosphatidylcholine; 2%-5% (w/v) glycerolor mannitol; 5% (w/v) albumin; 0.1 fM-10 mM histidine or cysteine; and apolar liquid carrier, wherein the lipid component and the amphiphilicemulsifier form free-moving lipid-carrying micelles (LMs) in the polarliquid carrier, wherein said LMs have an average diameter of 100 to 500nm and wherein the pharmaceutical composition is free of hemoglobin andfluorocarbon.

Another aspect of the present invention relates to a method for raisingblood pressure in a shock patent. The method comprises the step ofinfusing into said patient, an effective amount of a pharmaceuticalcomposition comprising a lipid component; an amphiphilic emulsifier; anda polar liquid carrier, wherein the lipid component and the amphiphilicemulsifier form free-moving lipid-carrying micelles (LM) in the polarliquid carrier, wherein the pharmaceutical composition is free ofhemoglobin and fluorocarbon, and wherein the pharmaceutical compositionis infused in an amount that equals to, or is greater than, 10% of thenormal blood volume of the shock patient at a rate greater than 200ml/min.

Another aspect of the present invention relates to a method for treatinga shock patent. The method comprises (a) infusing into said patient, aneffective amount of a first pharmaceutical composition comprising: afirst lipid component comprising soy bean oil; a first amphiphilicemulsifier; and a first polar liquid carrier, wherein the first lipidcomponent and the first amphiphilic emulsifier form free-movinglipid-carrying micelles (LM) in the first polar liquid carrier, whereinthe first pharmaceutical composition is free of hemoglobin andfluorocarbon; and (b) infusing into said patient, an effective amount ofa second pharmaceutical composition comprising: a second lipid componentcomprising chia bean oil; a second amphiphilic emulsifier; and a secondpolar liquid carrier, wherein the second lipid component and the secondamphiphilic emulsifier form free-moving LMs in the second polar liquidcarrier, wherein the second pharmaceutical composition is free ofhemoglobin and fluorocarbon.

BRIEF DESCRIPTION OF THE DRAWINGS

The detailed description will refer to the following drawings, whereinlike numerals refer to like elements, and wherein:

FIG. 1 is an illustration of a micelle containing a lipophilic core(yellow) and encapsulated by an emulsifier which may be a phospholipidor another amphiphilic molecule (mauve head black tails). The polarliquid carrier surrounds the micelle. These components together createan emulsion. The polar liquid may be water. This emulsion may be formedby adding energy to the mixture by sonication, using a homogenizer or amicrofluidizer.

FIG. 2 is a graph showing the linear relationship between micelleconcentration and oxygen content. Oxygen content was measured using massspectroscopy. The Y axis, designated “Carrying Capacity” is the ratio ofthe oxygen content of the emulsion over that of water in which bothfluids were exposed to the same conditions. The X axis is the of theemulsion comprised of micelles. The emulsion was prepared by sonicationof a mixture comprised of 1-3 grams of purified soybean oil, 0.1 gramsof soy lecithin and 0.24 grams of glycerol and water to bring themixture to a volume of 10 ml. Each point is the mean of 5 samples.

FIG. 3 is a diagram showing systolic blood pressure in mice treated withdifferent pharmaceutical compositions after severe hemorrhagic shock. LMis a commercially available model 20% soybean oil emulsion (Intralipid)alone. RL is Ringer's (L) lactate. The mean blood pressure immediatelybefore infusion across all experiments of [resuscitation] fluid was4.6+/−1.2 mmHg. The systolic pressure immediately before infusion of thefluid is subtracted out. Each point is the mean of 6-7 mice. **=p<0.01,*=p<0.05 for both the systolic and diastolic pressures of FIGS. 3 and 4,using a two tailed unpaired Student's t test.

FIG. 4 is a diagram showing diastolic blood pressure in mice treatedwith different pharmaceutical compositions after severe hemorrhagicshock. LM is a commercially available model 20% soybean oil emulsion(Intralipid) alone. RL is Ringer's (L) lactate. The mean blood pressureimmediately before infusion across all experiments was 4.6+/−1.2 mmHg.The diastolic pressure immediately before infusion of the fluid issubtracted out. Each point is the mean of 6-7 mice. P values are as withthe corresponding systolic pressure in FIG. 3.

FIG. 5 is a diagram showing the mean blood pressure after infusion as apercentage of the mean pre-hemorrhage blood pressure in mice treatedwith albumin-containing [resuscitation] fluids and mice treated withshed blood after severe hemorrhagic shock. The zero time point is thefirst pressure after initial infusion. There was no significantdifference between the pre-infusion pressures of any of the groups. Eachpoint is the mean of 6-7 mice. VS is the combination of 5% albumin with20% Intralipid. NSA is 5% albumin in 154 mM NaCL. RLA is 5% albumin inRinger's lactate. VS is significantly higher (p<0.05) than shed blood at5, 15 and 30 minutes using a two tailed, unpaired Student's test.

DETAILED DESCRIPTION

One aspect of the present invention relates to a pharmaceuticalcomposition for treating conditions related to lack of blood supply witha pharmaceutical composition. The pharmaceutical composition comprises alipophilic component, an emulsifier and a polar liquid carrier. Thelipophilic component is dispersed in the polar liquid carrier to form anemulsion that typically contains single layer micelles with a polarouter surface and an inner hydrophobic space filled with the lipophiliccomponent and/or other hydrophobic molecules. In certain embodiments,liposomes comprised of a lipid bilayer may also be employed. Thepharmaceutical composition can be used to increase blood pressure and tocarry oxygen to tissues in the absence of natural or modifiedhemoglobin. The pharmaceutical composition is free of hemoglobin,derivatives of hemoglobin, perfluorocarbon and derivatives ofperfluorocarbon.

As used herein, a composition is “free of hemoglobin, derivatives ofhemoglobin, perfluorocarbon and derivatives of perfluorocarbon” if thecomposition does not contain any hemoglobin, derivatives of hemoglobin,perfluorocarbon and derivatives of perfluorocarbon, or if thecomposition contains hemoglobin, derivatives of hemoglobin,perfluorocarbon and derivatives of perfluorocarbon at levels below 0.1%w/w.

As shown in FIG. 1, the lipid component and the emulsifier formlipid-containing micelles (LM) that are surrounded by the polar carrierfluid (shown as the white space around the amphiphilic molecules). Theamphiphilic emulsifier molecules occupy the periphery of the lipidboundary. The lipophilic ends of the amphiphilic emulsifier moleculesare directed inward toward the lipid and the polar ends of theamphiphilic emulsifier molecules are directed outward toward the polarcarrier fluid. Because hydrophobic gases, such as oxygen, preferentiallydissolve in the lipid core of the micelles relative to water or otheraqueous environments, the pharmaceutical composition of the presentinvention provides the ability to carry oxygen or other hydrophobicgases to body tissue. Emulsion comprised of the LM of the presentinvention is able to reverse the hypotension characteristic ofhemorrhagic shock, and absorb and release lipophilic gases such asoxygen and nitric oxide. The LMs in the pharmaceutical composition arealso capable of exerting an osmotic force and absorbing mediators ofvascular patency, such as prostaglandins, nitric oxide, leukotrienes,and hromboxane, and other lipophilic mediators such as plateletactivating factors.

In certain embodiments, the LM have diameters in the range of 10 to 5000nm. The colloidal properties of the LM with a diameter range from 10 to5000 nm promotes their retention in the intravascular space. In otherembodiments, the LM have diameters in the range of 100 to 500 nm. In yetother embodiments, the LM have diameters that are less than 100 nm(nanoemulsions). In shock of any etiology in which the capillarypermeability is increased, nanoemulsions would more readily get into theinterstitial space than larger structures. In certain embodiments, thenanoemulsion comprises a mixture of micelles of 2-300 nm in diameter. Inanother embodiment, the nanoemulsion comprises micelles with an averagediameter of about 1-30 nanometers (nano-micelles). At this size themicelle are able to get past the endothelial cell layer and enter theinterstitial space. The nano-micelles may be employed in situationswhere the permeability of the vascular space has not increased or topromote cellular absorption of lipophilic mediators or to promote entryof molecules or cellular components that can favorably modulateintracellular mechanisms. Such modulatory effects may also be effectedin the interstitial space especially in cases of increased vascular wallpermeability. For example, one of the mechanisms of shock fromhemorrhage or sepsis is the capillary leak. This capillary leak iscaused by the death of endothelial cells and the actions of neitrophils.It is mediated by cytokines such as IL-1 and TNF as well as nitricoxide. Neutrophils adhere to damaged endothelial cells and releasereactive oxygen species and cell wall damaging enzymes such asmyeloperoxidase. The nano-micelles could get into the interstitium viathe capillary leak and provide an anti inflammatory effect within theinterstitial space.

In some embodiments, the pharmaceutical composition comprises a mixtureof large micelles with diameters in the range of 100-400 nm andnano-micelles with diameters in the range of 1-30 nm. The largermicelles would tend to stay in the intravascular space and maintainintravascular volume while the nano-micelles could get into theinterstitium where they could favorably affect survival by blockingreactive oxygen species and delivering salutary molecules, such asinhibitors of apoptosis (e.g., Z-VAD-FMY, an apoptosis inhibitingpeptide) or protectors of mitochondrial integrity (e.g., Cyclosporin A,an inhibitor of mitochondrial inner pore opening). The nano-micelles mayalso be loaded with modulators of signal transduction such asdiacylglycerol or cyclic GMP, or antioxidant such as Coenzyme Q10. Incertain embodiments, the large micelles make up 10-40% (w/w) of thepharmaceutical composition, while the nano-micelles make up 5-30% (w/w)of the pharmaceutical composition. In other embodiments, the largemicelles (100-400 nm) are made of soybean oil and the nano-micelles(1-30 nm) are made of chia bean oil, which has a grater antiinflammatory effect than that of soybean oil.

The solubility of hydrophobic gases in the lipophilic core promotes theuptake and transport of these gases to tissues. The endogenouslyproduced gases carbon monoxide, nitric oxide and hydrogen sulfide canalso be carried in the emulsion for the modulation of the vascular toneand apoptotic processes. Oxygen may also be loaded for delivery totissues and the enhancement of aerobic metabolism. Xenon and argon arehydrophobic gases that could provide protection of the brain inhemorrhagic shock and in other pathological states such as seizures.Delivery of these gases to the brain may also provide pain relief.

In certain embodiments, the micelles in the pharmaceutical compositionof the present invention are free-moving micelles that are notencapsulated in any type of particles. Further, the wall of the micellesis comprised of either a single layer or a double layer of theamphiphilic emulsifier molecules so that the micelles may easily mergewith the cell membrane of the tissue that comes in contact with thepharmaceutical composition. Further, the micelles in the pharmaceuticalcomposition of the present invention are free of hemoglobin, derivativesof hemoglobin, perfluorocarbon and derivatives of perfluorocarbon.

The conditions related to lack of blood supply include, but are notlimited to, hypovolemia caused by bleeding, dehydration, vomiting,severe burns, systemic inflammatory response syndrome (SIRS) and drugssuch as diuretics or vasodilators. Severe hypovolemia may occur inconjunction with capillary leak (CL), which is present in differentconditions such as multiorgan dysfunction (MODS), sepsis, trauma, burn,hemorrhagic shock, post-cardiopulmonary bypass, pancreatitis andsystemic capillary leak syndrome, and causes morbidity and mortalityamong a large number of hospital patients.

Lipophilic Component

The lipophilic component can be any pharmaceutically acceptablelipophilic material, such as lipids. As shown in FIG. 1 the lipophiliccomponent is carried in the hydrophobic core of a micelle. Thehydrophobic core may also consist of additives that may enhance theuptake of hydrophobic gases such as branched molecules an example ofwhich is tri-n-octyl amine. In other embodiments, the lipophiliccomponent is trapped in the forms of structures, such as erythrocyteghosts.

As used herein, the term “lipid” refers to a fat-soluble material thatis naturally occurring, or non-naturally occurring. Examples of lipidsinclude but are not limited to, fatty acyls, glycerolipids,phospholipids, sphingolipids, sterol lipids, prenol lipids,saccharolipids, polyketides, non-natural lipid(s), cationic lipid(s),amphipathic alkyl amino acid derivative, adialkyldimethylammonium,polyglycerol alkyl ethers, polyoxyethylene alkyl ethers,tri-n-octylamine, boric acid, tris(3,5-dimethyl-4-heptyl)ester,triglycerides, diglycerides and other acylglycerols, such astetraglycerol, pentaglycerol, hexaglycerol, heptaglycerol, octoglycerol,nonaglyceol and decaglycerol, and mixtures thereof. In certainembodiments, the lipophilic component comprises soybean oil, chia beanoil or algae oil.

In one embodiment, the lipophilic component is soybean oil. Thelipophilic component may also be derived from chia beans that have ahigh concentration of anti-inflammatory omega 3 fatty acids. Soybean oilis thrombogenic and procoagulant, and therefore would be preferred forthe initial phase of hemorrhagic shock when clotting is desired. Afterbleeding is no longer an issue, oils rich in omega 3 fatty acids wouldbe favored because of their anti-thromobogenic properties. Oils rich inomega 3 fatty acids include, but are not limited to chia oil, algae oil,pumpkin oil, flaxseed oil or fish oil.

In certain embodiments, the lipid component comprises an unsaturatedfatty acid with one or more alkenyl functional groups in cis or transconfiguration. A cis configuration means that adjacent hydrogen atoms orother groups are on the same side of the double bond. In a transconfiguration theses moieties are on different sides of the double bond.The rigidity of the double bond freezes its conformation and, in thecase of the cis isomer, causes the chain to bend and restricts theconformational freedom of the fatty acid. In general, the more doublebonds the chain has, the less flexibility it has. When a chain has manycis bonds, it becomes quite curved in its most accessible conformations.For example, oleic acid, with one double bond, has a “kink” in it, whilelinoleic acid, with two double bonds, has a more pronounced bend.Alpha-linolenic acid, with three double bonds, favors a hooked shape.The effect of this is that in restricted environments, such as whenfatty acids are part of a phospholipid in a lipid bilayer, ortriglycerides in lipid droplets, cis bonds limit the ability of fattyacids to be closely packed and therefore could affect the meltingtemperature of the membrane or of the fat. In some embodiments, thelipid component comprises up to 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%(w/w) unsaturated fatty acid(s) that have one or more alkenyl functionalgroups in cis configuration.

Examples of cis-unsaturated fatty acids include, but are not limited to,obtusilic acid, linderic acid, tsuzuic acid, palmito-oleic acid, oleicacid, elaidic acid, vaccenic acid, petroselinic acid, gadoleic acid,eicosenoic acid, erucic acid, cetoleic acid, nervonic acid, ximenic acidand lumepueic acid; n-3 type unsaturated fatty acids such as α-linolenicacid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid,docosapentaenoic acid and docosahexaenoic acid; n-6 type unsaturatedfatty acids such as linoleic acid, linoelaidic acid, γ-linolenic acid,bis-homo-γ-linolenic acid and arachidonic acid; conjugated fatty acidssuch as conjugated linoleic acid and α-eleostearic acid; fatty acidscarrying double bonds at the 5-position thereof such as pinolenic acid,sciadonic acid, juniperic acid and columbinic acid; polyvalentunsaturated fatty acids, other than those listed above, such ashiragonic acid, moroctic acid, clupanodonic acid and nishinic acid;branched fatty acids such as isobutyric acid, isovaleric acid, iso acidand anti-iso acid; hydroxy fatty acids such as α-hydroxy acid, β-hydroxyacid, mycolic acid and polyhydroxy acid; epoxy-fatty acids; keto-fattyacids; and cyclic fatty acids. In certain embodiments, the lipophiliccomponent also comprises amphiphilic molecules.

The lipid component may constitute about 1-80%, 5-50% (w/v), 10-40%(w/v), or 10-30% (w/v) of the pharmaceutical composition. In certainembodiments, the lipid component constitutes about 10%, about 15%, about20%, about 25%, about 30% and about 35% (w/v) of the pharmaceuticalcomposition.

Emulsifier

The emulsifier can be any amphiphiles or amphiphilic molecule that willhave its hydrophobic tail in the lipophilic core of the micelle and itshydrophilic end in contact with the polar carrier. Examples ofemulsifiers are egg phospholipids, pure phospholipids, or amphiphilicpeptides.

As used herein, the term “amphiphiles” refers to a chemical compoundpossessing both hydrophilic and lipophilic properties. Examples ofamphiphiles include, but are not limited to, naturally-occurringamphiphiles such as phospholipids, cholesterol, glycolipids, fattyacids, bile acids, and saponins; and synthetic amphiphiles.

Examples of phospholipids include natural or synthetic phospholipidssuch as phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidic acid, phosphatidylglycerol,phosphatidylinositol, lisophosphatidylcholine, sphingomyelin, egg yolklecithin, soybean lecithin, and a hydrogenated phospholipid.

Examples of the glycolipids include glyceroglycolipids andsphingoglycolipids. Examples of glyceroglycolipids include digalactosyldiglycerides (such as digalactosyl dilauroyl glyceride, digalactosyldimyristoyl glyceride, digalactosyl dipalmitoyl glyceride, anddigalactosyl distearoyl glyceride) and galactosyl diglycerides (such asgalactosyl dilauroyl glyceride, galactosyl dimyristoyl glyceride,galactosyl dipalmitoyl glyceride, and galactosyl distearoyl glyceride).Examples of sphingoglycolipids include galactosyl cerebroside, lactosylcerebroside, and ganglioside.

Examples of the sterols include cholesterol, cholesterol hemisuccinate,3β-[N—(N′,N′-dimethylaminoethane)carbamoyl]cholesterol, ergosterol, andlanosterol.

In one embodiment, the emulsifier is egg phospholipids. In anotherembodiment, the emulsifier is soybean lecithin oralpha-phosphatidylcholine.

The emulsifier may constitute about 0.1-5% (w/v) of the pharmaceuticalcomposition. In certain embodiments, the lipid component constitutesabout 0.5%, about 0.75%, about 1%, about 1.2%, about 1.5% and about 2%(w/v) of the pharmaceutical composition.

Polar Liquid Carrier

The polar liquid carrier can be any pharmaceutically acceptable polarliquid that is capable of forming an emulsion with the lipid. The term“pharmaceutically acceptable” refers to molecular entities andcompositions that are of sufficient purity and quality for use in theformulation of a composition or medicament of the present invention andthat, when appropriately administered to an animal or a human, do notproduce an adverse, allergic or other untoward reaction. Since bothhuman use (clinical and over-the-counter) and veterinary use are equallyincluded within the scope of the present invention, a pharmaceuticallyacceptable formulation would include a composition or medicament foreither human or veterinary use. In one embodiment, the polar liquidcarrier is water or a water based solution. In another embodiment, thepolar liquid carrier is a non-aqueous polar liquid such as dimethylsulfoxide, polyethylene glycol and polar silicone liquids.

A water-based solution generally comprises a physiologically compatibleelectrolyte vehicle isosmotic or near isosmotic with whole blood. Thecarrier can be, for example, physiological saline, a saline-glucosemixture, Ringer's solution, lactated Ringer's solution, Locke-Ringer'ssolution, Krebs-Ringer's solution, Hartmann's balanced saline,heparinized sodium citrate-citric acid-dextrose solution, and polymericplasma substitutes, such as polyethylene oxide, polyvinyl pyrrolidone,polyvinyl alcohol and ethylene oxide-propylene glycol condensates. Thepharmaceutical composition may additionally comprise other constituentssuch as pharmaceutically-acceptable carriers, diluents, fillers andsalts, the selection of which depends on the dosage form utilized, thecondition being treated, the particular purpose to be achieved accordingto the determination of the ordinarily skilled artisan in the field andthe properties of such additives.

Gas Carrying Capacity of the Emulsion

The lipid component in the emulsion, in forms such as micelles and/orerythrocyte ghosts provides the ability for the emulsion to carry alarger amount of lipophilic gases than of a purely aqueous solution.Specifically, the lipophilic gases are dissolved into the lipophilicportion of the emulsion to form a homogeneous solution with the lipidcomponent and any other hydrophobic liquid material that may be presentin the lipophilic portion of the emulsion.

In one embodiment, the lipophilic gas is oxygen. Oxygen is 4.41 timesmore soluble in lipid than in water (Battion et al., J. Amer. Oil Chem.Soc. 1968, 45:830-833). Accordingly, an emulsion with a higher lipidcontent would be able to carry more oxygen than an emulsion with alesser lipid content. In one embodiment, the emulsion has a lipidcontent of about 1-80% (w/v). In other embodiments, the emulsion has alipid content of about 10-80% (w/v), 20-60% (w/v), about 20-50% (w/v),about 20-40% (w/v) or about 20-25% (w/v). In yet another embodiment, theemulsion has a lipid content of about 21.8%. In certain embodiments, theemulsion is prepared by mixing the lipid component and the polar liquidcomponent in the presence of regular air. In other embodiment, theemulsion is further oxygenated by bubbling regular air or pure oxygenthrough the emulsion for a desired period of time. Since bubbles areundesirable in the circulation due to the possibility of airembolization a bubble trap would have to be added to remove bubblesleaving only the gas that has been solubilized in the core of themicelle, in the polar carrier or attached to proteins or otheradditives. The gas may also be loaded onto the micelles by equilibrationof the micelles with an atmosphere enriched with the gas combined withgentle movement of the emulsion in a mixture chamber in order to avoidthe creation of bubbles. Loading may also be done under pressuresgreater than 1 atmosphere followed by release of the pressure to allowthe release of excess gas.

In another embodiment, the lipophilic gas is xenon (Xe) or argon (Ar).In another embodiment, the lipophilic gas is nitric oxide (NO). Inanother embodiment, the lipophilic gas is hydrogen sulfide (H₂S). In yetanother embodiment, the lipophilic gas is carbon monoxide (CO).

In one embodiment, the pharmaceutical composition contains micellesloaded with a gas mixture (e.g., a mixture of oxygen, hydrogen sulfide,carbon monoxide and/or nitric oxide). In another embodiment, the[resuscitation] pharmaceutical composition contains a mixture ofmicelles loaded with various gases. For example, the mixture of micellesmay contain 50% NO-loaded micelles and 50% O₂-loaded micelles.

Rigid Nonplanar Molecules

The [resuscitation] pharmaceutical composition may further comprisemolecules with a rigid nonplanar structure. Such molecules will creategreater irregularity and more space for gas molecules in the hydrophobiccore of the micelle structure, thereby modifying the gas carryingcapacity of the micelles. Examples of such molecules include, but arenot limited to, (+) naloxone, (+) morphine, and (+) naltrexone.

In one embodiment, molecules with a rigid nonplanar structure is (+)naloxone which, unlike the opiate receptor antagonist (−) naloxone, doesnot bind to opiate receptor and will not increase pain as (−) naloxonewould. In another embodiment, (+) naloxone is used at a concentration of10⁻⁵-10⁻⁴ M. In another embodiment, (+) naloxone is used at aconcentration of 10⁻⁴ M or higher.

Upon resuscitation, an inflammatory process may be triggered inreperfused tissues (ischemic—reperfusion injury) causing endothelialcell (EC) injury and capillary leak (CL). In sepsis and other diseases,systemic inflammation may be triggered by the disease and in a similarsequence leads to EC injury, CL, and ultimately hypovolemic shock thatrequires resuscitation. Accordingly, in one embodiment, (+) naloxone isused at a concentration range that produces anti-inflammatory effect at10⁻⁵-10⁻⁴M (Simpkins C O, Ives N, Tate E, Johnson M. Naloxone inhibitssuperoxide release from human neutrophils. Life Sci. 1985 Oct. 14;37(15):1381-6)

Molecules with a nonplanar structure also include organic molecules withbranched structures. Examples of such molecules include, but are notlimited to, tri-n-octylamine, tri-n-hexylamine, boric acid,tris(3,5,-dimethyl-4-heptyl)ester, metal complexed and non-metalcomplexed deuteroporphyrin dimethyl esters and their derivatives,hexaphenylsilole, and silicone polymers.

Plasma Component

The pharmaceutical composition may further comprise a plasma component.In one embodiment, the plasma is an animal plasma. In anotherembodiment, the plasma is human plasma. Although not wishing to be boundby any particular scientific theory, it is believed that theadministration of blood substitutes may dilute the concentration ofcoagulation factors to an undesirable level. Accordingly, using plasmaas the diluent for the oxygen carrying component avoids this problem.Plasma can be collected by any means known in the art, provided that redcells, white cells and platelets are essentially removed. Preferably, itis obtained using an automated plasmaphoresis apparatus. Plasmaphoresisapparatuses are commercially available and include, for example,apparatuses that separate plasma from the blood by ultrafiltration or bycentrifugation. An ultrafiltration-based plasmaphoresis apparatus suchas manufactured by Auto C, A200 (Baxter International Inc., Deerfield,Ill.) is suitable because it effectively removes red cells, white cellsand platelets while preserving coagulation factors.

Plasma may be collected with an anticoagulant, many of which are wellknown in the art. Preferred anti-coagulants are those that chelatecalcium such as citrate. In one embodiment, sodium citrate is used as ananticoagulant at a final concentration of 0.2-0.5%, preferably 0.3-0.4%,and most preferably at 0.38%. The plasma may be fresh, frozen, pooledand/or sterilized. While plasma from exogenous sources may be preferred,it is also within the present invention to use autologous plasma that iscollected from the subject prior to formulation and administration ofthe pharmaceutical composition.

In addition to plasma from natural sources, synthetic plasma may also beused. The term “synthetic plasma,” as used herein, refers to any aqueoussolution that comprises at least one plasma protein. Proteins resemblingplasma protein may also be used.

Oncotic Agent

In one embodiment, the pharmaceutical composition further contains anoncotic agent in addition to the lipid micelles. The oncotic agent iscomprised of molecules whose size is sufficient to prevent their lossfrom the circulation by traversing the fenestrations of the capillarybed into the interstitial spaces of the tissues of the body. Examples ofoncotic agents include, but are not limited to, dextran (e.g., alow-molecular-weight dextran), dextran derivatives (e.g., carboxymethyldextran, carboxydextran, cationic dextran, and dextran sulfate),hydroxyethyl starch, hydroxypropyl starch, branched, unsubstituted orsubstituted starch, gelatin (e.g., modified gelatin), albumin (e.g.,human plasma, human serum albumin, heated human plasma protein, andrecombinant human serum albumin), PEG, polyvinyl pyrrolidone,carboxymethylcellulose, acacia gum, glucose, a dextrose (e.g., glucosemonohydrate), oligosaccharides (e.g., oligosaccharide), a polysaccharidedegradation product, an amino acid, and a protein degradation product.Among those, particularly preferable are low-molecular-weight dextran,hydroxyethyl starch, modified gelatin, and recombinant albumin.

Because of its antioxidant effects, albumin may also be used to minimizereactive oxygen species interaction with the components of the micelleand may also stabilize the micelle structure. In one embodiment, the.oncotic agent is about 2%, 5%, 7% or 10% (w/v) albumin. In anotherembodiment, the oncotic agent is a polysaccharide, such as Dextran, in amolecular weight range of 30,000 to 50,000 daltons (D). In yet anotherembodiment, the oncotic agent is a polysaccharide, such as Dextran, in amolecular weight range of 50,000 to 70,000 D. High molecular weightdextran solutions are more effective in preventing tissue swelling dueto their lower rates of leakage from capillaries.

In one embodiment, the concentration of the polysaccharide is sufficientto achieve (when taken together with chloride salts of sodium, calciumand magnesium, organic ion from the organic salt of sodium and hexosesugar discussed above) colloid osmotic pressure approximating that ofnormal human serum, about 28 mm Hg.

In another embodiment the oncotic agent is glycerol or mannitol in anamount of 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% up to 30% (w/v) of thepharmaceutical composition. In other embodiments, pharmaceuticalcomposition comprises glycerol or mannitol in an amount of 2-5% w/v.

Crystalloid Agent

The pharmaceutical composition may also comprise a crystalloid agent.The crystalloid agent can be any crystalloid which, in the form of thepharmaceutical composition composition, is preferably capable ofachieving an osmolarity greater than 800 mOsm/l, i.e. it makes thepharmaceutical composition “hypertonic”. Examples of suitablecrystalloids and their concentrations in the pharmaceutical compositioninclude, but are not limited to, 3% w/v NaCl, 7% NaCl, 7.5% NaCl, and7.5% NaCl in 6% w/v dextran. In one embodiment, the [resuscitation]pharmaceutical composition has an osmolarity of between 800 and 2400mOsm/l.

When the pharmaceutical composition further comprises a crystalloid andis hypertonic, the pharmaceutical composition may provide improvedfunctionality for rapid recovery of hemodynamic parameters over othercompositions, which include a colloid component. Small volume highlyhypertonic crystalloid infusion (e.g., 1-10 ml/kg) provides significantbenefits in the rapid and sustained recovery of acceptable hemodynamicparameters in controlled hemorrhage. In another embodiment, the lipidemulsion used is Intralipid®. In another embodiment, the lipid emulsionused is 20% Intralipid®. In one embodiment, the lipid comprisesanti-inflammatory lipids such as omega-3 fatty acids. Hypertonicity mayalso be achieved by adding glycerol.

Anti-Inflammatory and Immunomodulatory Agent

In one embodiment, the pharmaceutical composition of the presentinvention further includes an anti-inflammatory or immunomodulatoryagent. Examples of the anti-inflammatory agent shown to inhibit reactiveoxygen species including, but are not limited to, histidine, albumin,(+) naloxone, prostaglandin D₂, molecules of the phenylalkylamine class.Other anti-inflammatory compounds and immunomodulatory drug includeinterferon; interferon derivatives comprising betaseron, β-interferon;prostane derivatives comprising iloprost, cicaprost; glucocorticoidscomprising cortisol, prednisolone, methyl-prednisolone, dexamethasone;immunsuppressives comprising cyclosporine A, methoxsalene,sulfasalazine, azathioprine, methotrexate; lipoxygenase inhibitorscomprising zileutone, MK-886, WY-50295, SC-45662, SC-41661A, BI-L-357;leukotriene antagonists; peptide derivatives comprising ACTH and analogsthereof; soluble TNF-receptors; anti-TNF-antibodies; soluble receptorsof interleukins or other cytokines; antibodies against receptors ofinterleukins or other cytokines, T-cell-proteins; and calcipotriols andanalogues thereof taken either alone or in combination.

Electrolytes

In one embodiment, the [resuscitation] pharmaceutical composition of thepresent invention includes one or more electrolytes. The electrolyte tobe used in the present invention typically includes various electrolytesto be used for medicinal purposes. Examples of the electrolyte includesodium salts (e.g., sodium chloride, sodium hydrogen carbonate, sodiumcitrate, sodium lactate, sodium sulfate, sodium dihydrogen phosphate,disodium hydrogen phosphate, sodium acetate, sodium glycerophosphate,sodium carbonate, an amino acid sodium salt, sodium propionate, sodiumβ-hydroxybutyrate, and sodium gluconate), potassium salts (e.g.,potassium chloride, potassium acetate, potassium gluconate, potassiumhydrogen carbonate, potassium glycerophosphate, potassium sulfate,potassium lactate, potassium iodide, potassium dihydrogen phosphate,dipotassium hydrogen phosphate, potassium citrate, an amino acidpotassium salt, potassium propionate, and potassium β-hydroxybutyrate),calcium salts (e.g., calcium chloride, calcium gluconate, calciumlactate, calcium glycerophosphate, calcium pantothenate, and calciumacetate), magnesium salts (e.g., magnesium chloride, magnesium sulfate,magnesium glycerophosphate, magnesium acetate, magnesium lactate, and anamino acid magnesium salt), ammonium salts (e.g., ammonium chloride),zinc salts (e.g., zinc sulfate, zinc chloride, zinc gluconate, zinclactate, and zinc acetate), iron salts (e.g., iron sulfate, ironchloride, and iron gluconate), copper salts (e.g., copper sulfate), andmanganese salts (for example, manganese sulfate). Among those,particularly preferable are sodium chloride, potassium chloride,magnesium chloride, disodium hydrogen phosphate, dipotassium hydrogenphosphate, potassium dihydrogen phosphate, sodium lactate, sodiumacetate, sodium citrate, potassium acetate, potassium glycerophosphate,calcium gluconate, calcium chloride, magnesium sulfate, and zincsulfate.

Concentration of calcium, sodium, magnesium and potassium ion istypically within the range of normal physiological concentrations ofsaid ions in plasma. In general, the desired concentration of these ionsis obtained from the dissolved chloride salts of calcium, sodium andmagnesium. The sodium ions may also come from a dissolved organic saltof sodium that is also in solution.

In one embodiment, the sodium ion concentration is in a range from 70 mMto about 160 mM. In another embodiment, the sodium ion concentration isin a range of about 130 to 150 mM.

In one embodiment, the concentration of calcium ion is in a range ofabout 0.5 mM to 4.0 mM. In another embodiment, the concentration ofcalcium ion is in a range of about 2.0 mM to 2.5 mM.

In one embodiment, the concentration of magnesium ion is in a range of 0to 10 mM. In another embodiment, the concentration of magnesium ion isin a range of about 0.3 mM to 0.45 mM. It is best not to includeexcessive amounts of magnesium ion in the [resuscitation] pharmaceuticalcomposition of the invention because high magnesium ion concentrationsnegatively affect the strength of cardiac contractile activity. In apreferred embodiment of the invention, the solution containssubphysiological amounts of magnesium ion.

In one embodiment, the concentration of potassium ion is in asubphysiological range of between 0-5 mEq/l K⁺ (0-5 mM), preferably 2-3mEq/l K⁺ (2-3 mM). Thus, the pharmaceutical composition allows fordilution of the potassium ion concentration in stored transfused blood.As a result, high concentrations of potassium ion and potential cardiacarrhythmias and cardiac insufficiency caused thereby can be more easilycontrolled. The pharmaceutical composition containing a subphysiologicalamount of potassium is also useful for purposes of blood substitutionand low temperature maintenance of a subject.

In one embodiment, the concentration of chloride ion is in the range of70 mM to 160 mM. In another embodiment, the concentration of chlorideion is in the range of 110 mM to 125 mM.

Other sources of ions include sodium salts (e.g., sodium hydrogencarbonate, sodium citrate, sodium lactate, sodium sulfate, sodiumdihydrogen phosphate, disodium hydrogen phosphate, sodium acetate,sodium glycerophosphate, sodium carbonate, an amino acid sodium salt,sodium propionate, sodium β-hydroxybutyrate, and sodium gluconate),potassium salts (e.g., potassium acetate, potassium gluconate, potassiumhydrogen carbonate, potassium glycerophosphate, potassium sulfate,potassium lactate, potassium iodide, potassium dihydrogen phosphate,dipotassium hydrogen phosphate, potassium citrate, an amino acidpotassium salt, potassium propionate, and potassium β-hydroxybutyrate),calcium salts (e.g., calcium gluconate, calcium lactate, calciumglycerophosphate, calcium pantothenate, and calcium acetate), magnesiumsalts (e.g., magnesium sulfate, magnesium glycerophosphate, magnesiumacetate, magnesium lactate, and an amino acid magnesium salt), ammoniumsalts, zinc salts (e.g., zinc sulfate, zinc chloride, zinc gluconate,zinc lactate, and zinc acetate), iron salts (e.g., iron sulfate, ironchloride, and iron gluconate), copper salts (e.g., copper sulfate), andmanganese salts (for example, manganese sulfate). Among those,particularly preferable are sodium chloride, potassium chloride,magnesium chloride, disodium hydrogen phosphate, dipotassium hydrogenphosphate, potassium dihydrogen phosphate, sodium lactate, sodiumacetate, sodium citrate, potassium acetate, potassium glycerophosphate,calcium gluconate, calcium chloride, magnesium sulfate, choline chlorideand zinc sulfate.

Carbohydrates and Amino Acids

The pharmaceutical composition may contain a carbohydrate or a mixtureof carbohydrates. Suitable carbohydrates include, but are not limitedto, simple hexose (e.g., glucose, fructose and galactose), mannitol,sorbitol or others known to the art. In one embodiment, thepharmaceutical composition includes physiological levels of a hexose.“Physiological levels of a hexose” includes a hexose concentration ofbetween 2 mM to 50 mM. In one embodiment, the pharmaceutical compositioncontains 5 mM glucose. At times, it is desirable to increase theconcentration of hexose in order to provide nutrition to cells. Thus therange of hexose may be expanded up to about 50 mM if necessary toprovide minimal calories for nutrition.

Other suitable carbohydrates include various saccharides to be used formedicinal purposes. Examples of the saccharides include xylitol,dextrin, glycerin, sucrose, trehalose, glycerol, maltose, lactose, anderythritol.

The pharmaceutical composition may contain an amino acid or a mixture ofamino acids. Suitable amino acids include, but are not limited to,alanine, arginine, aspartate, asparagine, cysteine, glutamate,glutamine, glycine, histidine, isoleuc leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine,threonine, tryptophan, valine and 2-aminopentaenoic acid. In oneembodiment, the amino acid is selected from the group consisting ofhistidine, tyrosine, phenylalanine and cysteine. In another embodiment,the pharmaceutical composition comprises an amino acids known to preventapoptosis. Examples of such amino acids include glutamine, glycine,proline and 2-aminopentaenoic acid.

The amino acid may be used in the concentration range of 0.1 fM-200 mM,0.1 fM-100 pM, 100 pM-10 nM, 10 nM-10 μM, 0.01-200 mM, 0.2-50 mM, or0.5-2 mM. In one embodiment, the amino acid is used at a concentrationof 1 mM.

Buffering Agent

The pharmaceutical composition of the present invention may furthercomprise a biological buffer to maintain the pH of the fluid at thephysiological range of pH7-8. Examples of biological buffers include,but are not limited to,N-2-Hydroxyethylpiperazine-N′-2-hydroxypropanesulfonic acid (HEPES),3-(N-Morpholino)propanesulfonic acid (MOPS),2-([2-Hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)glyci ethanesulfonicacid (TES),3-[N-tris(Hydroxy-methyl)methylamino]-2-hydroxyethyl]-1-piperazinepropanesulfonic acid (EPPS), Tris[hydrolymethyl]-aminoethane (THAM), andTris[Hydroxylmethyl]methyl aminomethane (TRIS).

In one embodiment, the buffering agent is histidine, imidazole,substituted histidine or imidazole compounds retaining the amphotericsite of the imidazole ring, oligopeptides containing histidine, orglycyglycine or mixtures thereof. Histidine is also capable of reducingreactive oxygen species and inhibiting cell shrinkage. (see e.g.,Simpkins et al., J. Trauma. 2007, 63:565-572). Histidine or imidazolemay be used in a concentration range of about 0.0001M to about 0.2M,preferably about 0.0001M to about 0.01M. In one embodiment, histidine isused at a concentration of about 1 mM.

In another embodiment, the pharmaceutical composition of the presentinvention uses normal biological components to maintain in vivobiological pH. Briefly, some biological compounds, such as lactate, arecapable of being metabolized in vivo and act with other biologicalcomponents to maintain a biologically appropriate pH in an animal. Thebiological components are effective in maintaining a biologicallyappropriate pH even at hypothermic temperatures and at essentiallybloodless conditions. Examples of the normal biological componentsinclude, but are not limited to carboxylic acids, salt and esterthereof. Carboxylic acids have the general structural formula of RCOOX,where R is an alkyl, alkenyl, or aryl, branched or straight chained,containing 1 to 30 carbons which carbons may be substituted, and X ishydrogen or sodium or other biologically compatible ion substituentwhich can attach at the oxygen position, or is a short straight orbranched chain alkyl containing 1-4 carbons, e.g., —CH₃, —CH₂CH₃.Examples of carboxylic acids and carboxylic acid salts include, but arenot limited to, lactate and sodium lactate, citrate and sodium citrate,gluconate and sodium gluconate, pyruvate and sodium pyruvate, succinateand sodium succinate, and acetate and sodium acetate.

Coagulation Enhancers

Aggressive high volume resuscitation, without controlling the bleeding,can exacerbate the hemorrhage by disrupting the early formed softthrombi, and by diluting coagulation factors. In certain embodiments,the pharmaceutical composition may further comprise one or morecoagulation enhancers. Examples of coagulation factors include, but arenot limited to, factor 7, thrombin and platelets. These factors may befrom natural or non-natural sources. In certain embodiments, factor 7 isadded to the pharmaceutical composition at a concentration of 70-150IU/kg, prothrombin complex is added to the pharmaceutical composition ata concentration of 15-40 IU/kg, and fibrinogen is added to the[resuscitation] pharmaceutical composition at a concentration of 50-90mg/kg. Naturally-derived or synthetic platelets or platelet substitutesmay also be added.

Antioxidants

In certain embodiments, the pharmaceutical composition may furthercomprise one or more antioxidants. Examples of antioxidants include, butare not limited to, sodium hydrogen sulfite, sodium sulfite, sodiumpyrosulfite (e.g., sodium metabisulfite), rongalite (CH2OHSO2Na),ascorbic acid, sodium ascorbate, erythorbic acid, sodium erythorbate,cysteine, cysteine hydrochloride, homocysteine, glutathione,thioglycerol, α-thioglycerin, sodium edetate, citric acid, isopropylcitrate, potassium dichloroisocyanurate, sodium thioglycolate, sodiumpyrosulfite 1,3-butylene glycol, disodium calciumethylenediaminetetraacetate, disodium ethylenediaminetetraacetate, anamino acid sulfite (e.g, L-lysine sulfite), butylhydroxyanisole (BHA),butylhydroxytoluene (BHT), propyl gallate, ascorbyl palmitate, vitamin Eand derivatives thereof (e.g., dl-α-tocopherol, tocopherol acetate,natural vitamin E, d-δ-tocopherol, mixed tocopherol, and trolox),guaiac, nordihydroguaiaretic acid (NDGA), L-ascorbate stearate esters,soybean lecithin, palmitic acid ascorbic acid, benzotriazol, andpentaerythrityl-tetrakis[3-(3,5-di-t-butyl-4-hydroxyphenyepropionate]2-mercaptobenzimidazole.Among those, preferable are sodium hydrogen sulfite, sodium sulfite,ascorbic acid, homocysteine, dl-α-tocopherol, tocopherol acetate,glutathione, and trolox.

Other Components

In addition to the components discussed above, the pharmaceuticalcomposition may further comprise other additives that include, but arenot limited to, antibiotics, such as penicillin, cloxacillin,dicloxacillin, cephalosporin, erythromycin, amoxicillin-clavulanate,ampicillin, tetracycline, trimethoprim-sulfamethoxazole,chloramphenicol, ciprofloxacin, aminoglycoside (e.g., tobramycin andgentamicin), streptomycin, sulfa drugs, kanamycin, neomycin, landmonobactams; anti-viral agents, such as amantadine hydrochloride,rimantadin, acyclovir, famciclovir, foscarnet, ganciclovir sodium,idoxuridine, ribavirin, sorivudine, trifluridine, valacyclovir,valgancyclovir, pencyclovir, vidarabin, didanosine, stavudine,zalcitabine, zidovudine, interferon alpha, and edoxudine; anti-fungalagents such as terbinafine hydrochloride, nystatin, amphotericin B,griseofulvin, ketoconazole, miconazole nitrate, flucytosine,fluconazole, itraconazole, clotrimazole, benzoic acid, salicylic acid,voriconazole, caspofungin, and selenium sulfide; vitamins, amino acids,vessel expanders such as alcohols and polyalcohols, surfactants,antibodies against harmful cytokines such as tumor necrosis factor (TNF)or interleukins, and mediators of vascular potency and immunomoduators,such as prostaglandins, leukotrienes, pro-opiomelanocortin fragments andplatelet activating factors.

In certain embodiments, the pharmaceutical composition may furthercontain beneficial anions such as lactate or glutamate. Hypertoniclactate containing compositions have been found to be effective inreducing brain edema in patients with acute hemodynamic distress. In oneembodiment, the pharmaceutical composition contains 250 to 2400 mM oflactic acid or lactate. In another embodiment, the pharmaceuticalcomposition contains 250 to 2400 mM of lactic acid or lactate and 2 to10 mM potassium.

In certain other embodiments, the pharmaceutical composition may containsubstituted cations. For example, the pharmaceutical composition maycontain choline to substitute sodium ions.

In certain embodiments, the pharmaceutical composition further containsanti-cancer drugs and/or intracellular signal molecules, such as cAMPand diacylglycerol. In other embodiments, the pharmaceutical compositionfurther contain one or more organelles or organelle components such asendoplasmic reticulum, ribosomes, and mitochondria in whole or in part.

In other embodiments, the pharmaceutical composition may be combinedwith red blood cells, modified red blood cells or other cellularcomponents of blood.

In yet other embodiments, the pharmaceutical composition furthercomprises proopiomelanocortin fragments, such as beta endorphin,melonocyte stimulating hormone enkephalins or opiates to modify theimmune response and to provide analgesia. Beta endorphin may also beused at a final concentration of 0.01-100 nm, preferably 0.1-10 nm, morepreferably about 1 nm, to modulate neutrophilic function in the septicstate (See, e.g., Simpkins et al., J Natl Med. Assoc. 1988, 80:199-203).

In yet other embodiments, the pharmaceutical composition furthercomprises one or more neurotropic agents for treatment of psychiatricdisease or prevention of psychiatric disease.

The pharmaceutical composition possesses the ability to absorb toxicchemical molecules/biomolecules produced as the result of trauma orhemorrhagic shock. For example, lymph factors produced in gut andthoracic duct lymph nodes may result in acute lung injury and red bloodcell deformability after trauma/hemorrhagic shock. Other toxic chemicalmolecules/biomolecules include, but are not limited to, leukotrienes,prostaglandins, nitric oxide, endotoxin and tumor necrosis factor (TNF).The lipid emulsion in the pharmaceutical composition allows effectiveabsorption of lipophilic chemical molecules/biomolecules. In certainembodiment, the pharmaceutical composition further contains antagoniststo toxic chemical molecules/biomolecules, such as antibodies toendotoxins.

In one embodiment, the pharmaceutical composition comprises 10-50% (w/v)soybean oil, 1-1.5% (w/v) soybean lecithin and 1-1.5% (w/v) glycerol. Inanother embodiment, the pharmaceutical composition further comprises10-30% (w/v) soybean oil, 1-1.5% (w/v) soybean lecithin, 1-1.5% (w/v)glycerol, 102 mM NaCl, 28 mM Na(L) lactate, and 4 mM KCL. In anotherembodiment, the pharmaceutical composition further comprises 10-30%(w/v) soybean oil, 1-1.5% (w/v) soybean lecithin, 1-1.5% (w/v) glycerol,102 mM NaCl, 28 mM Na(L) lactate, 4 mM KCL and 1 mM histidine, tyrosine,phenylalanine or cysteine. The pharmaceutical composition is preparedunder conditions that form micelles with an average diameter of 5 μm orsmaller. In one embodiment, the micelles in the pharmaceuticalcomposition have an average diameter of 1 μm or smaller. In anotherembodiment, the micelles in the pharmaceutical composition have anaverage diameter of 0.3 μm or smaller. In another embodiment, themicelles in the pharmaceutical composition have an average diameter of0.1 μm or smaller. In another embodiment, the micelles in thepharmaceutical composition have an average diameter of 0.03 μm orsmaller. In yet another embodiment, the micelles in the pharmaceuticalcomposition have an average diameter of 0.01 μm or smaller. In anotherembodiment the hydrophobic component is carried within erythrocyteghosts. Other oils such as oil from chia beans, pumpkin seeds or othersources may be used to take advantage of their ability to modulatenegatively or positively the processes of coagulation, proliferation,immune response or nutrition as needed by the disease process beingaddressed. In certain embodiments, the above described pharmaceuticalcomposition further comprise about 2-40% (w/v), about 2-20% (w/v), about4-10% (w/v), or about 5% (w/v) albumin or albumin polymers or albuminpolymers conjugated with amino acids or peptides, which are added to thepharmaceutical composition after the formation of micelles.

In certain embodiments the pharmaceutical composition of the presentinvention comprises a lipid component selected from the group consistingof soybean oil, chia bean oil and algae oil, an emulsifier selected fromthe group consisting of phospholipids and α-phosphatidylcholine, and anamino acid or n-acetyl amino acid at a final concentration of 0.2-20 mM,0.5-10 mM, 0.5-5 mM or 0.5-2 mM. In certain embodiments, thepharmaceutical composition has an final amino acid concentration of 0.1,0.2, 0.5, 1, 2.5, 5, 7.5, or 10 mM. The emulsifier:the lipid componentratio (w/w) is about 1:400 to about 1:20, preferably about 1:200 toabout 1:50. In one embodiment, the emulsifier:the lipid component ratio(w/w) is about 1:100. In another embodiment, the emulsifier:the lipidcomponent ratio (w/w) is about 1.2:100.

The pharmaceutical composition is free of Ca⁺⁺, K⁺, and Mg⁺⁺ and Al⁺⁺⁺,as well as hemoglobin, derivatives of hemoglobin, perfluorocarbon andderivatives of perfluorocarbon. In certain embodiments, Ca⁺⁺ and K⁺ areadded to the pharmaceutical composition just prior to use (e.g., within24 hours prior to use). Because Al⁺⁺⁺ is toxic to bone, brain,hematopoiesis, heme synthesis, globulin synthesis, iron absorption andmetabolism, and fetal development, all oils and other components musthave the minimum amount of Al⁺⁺⁺ possible. In certain embodiments, thepharmaceutical composition contains Al⁺⁺⁺ at a concentration of lessthan 25 mg/l, 20 mg/l, 10 mg/l or 5 mg/l. In other embodiments, thepharmaceutical composition is free of Al⁺⁺⁺, i.e., undetectable byconventional methods.

In one embodiment, the pharmaceutical composition comprises soybean oil,egg yolk phospholipids and an amino acid, beta-endorphin or othermodulator that acts at femtomolar concentrations or higher at a finalconcentration of 0.1 femtomolar (fM) to 10 mM.

Preparation of the Pharmaceutical Composition

The pharmaceutical composition may be prepared by mixing the lipidcomponent, the emulsifier, the aqueous carrier, and any other componentsto form an emulsion. Commonly used mixing methods include, but are notlimited to, stirring, shaking, homogenization, vibration,microfluidization and sonication. In one embodiment, the pharmaceuticalcomposition is formed by mixing a pre-formed lipid emulsion, such asIntralipid® or Liposyn® with the aqueous carrier and other components.In addition, the pharmaceutical composition can be carried inerythrocyte ghosts. Specifically, the emulsion should be prepared inmanners that allow the lipophilic gases dissolving into the lipophilicportion of the emulsion but not forming microbubbles which may increasethe risk of gas embolization.

In certain embodiments, albumin or albumin polymers or albumin polymersconjugated with amino acids or peptides is added to the pharmaceuticalcomposition in an amount of 2-40% (w/v), about 2-20% (w/v), about 4-10%(w/v), or about 5% (w/v). The albumin or albumin polymers or albuminpolymers conjugated with amino acids or peptides is added to thepharmaceutical composition after the formation of micelles. In oneembodiment, the lipid component, the emulsifier, the aqueous carrier andany other non-albumin components are mixed to form an emulsion. Albumin,albumin polymers or albumin polymers conjugated with amino acids orpeptides is then dissolved in the emulsion at the desired concentration.

In one embodiment, the pharmaceutical composition is prepared accordingto the following recipes:

Recipe 1

Part A

soybean oil 20%=20 grams

glycerol 2.25%=2.25 grams

egg yolk phospholipids 2.4%=2.4 grams

add water to final volume of 100 ml

add sodium hydroxide until pH=8.0

stir or sonicate to produce micelles with average diameter of 300-400 nm

Part B

NaCl 102 mM=0.6 grams

Na (L) lactate 28 mM=0.31 grams

KCl 4 mM=0.03 grams

CaCl₂ 1.5 mM=0.06 grams

Human serum albumin 5%=5 grams

Part A may be used alone, or mixed with Part B within 24 hours of use.

Recipe 2

Part A

Soybean oil 20%=20 grams

Glycerol 2.25%=2.25 grams

Egg yolk phospholipids 2.4%=2.4 grams add water

to final volume of 100 ml

add sodium hydroxide until pH=8.0

stir or sonicate to produce micelles with average diameter of 300-400 nm

Part B

NaCl 102 mM=0.6 grams

Na (L) lactate 28 mM=0.31 grams

KCl 4 mM=0.03 grams

CaCl2 1.5 mM=0.06 grams

1 mM amino acid histidine or cysteine

β-endorphin 1 nM

Part A may be used alone, or mixed with Part B within 24 hours of use.

In some embodiments, Part A or the mixture of Part A and Part B, isloaded with oxygen, nitric oxide, carbon monoxide, xenon, argon,hydrogen sulfide other hydrophobic gases or mixtures of these gasesprior to use. These gases may be used in shock to deliver oxygen foraerobic metabolism after the initial bolus, provide an initial carbonmonoxide bolus to protect against reperfusion injury, to open vessels invascular diseases or states involving vascular constriction orobstruction, xenon or argon to protect against the effects of traumaticbrain injury or seizures, or hydrogen sulfide to promote long-termtissue preservation. Nitric oxide loaded micelles may also be used as ananti-hypertensive medication. Either Part A, Part B, or the mixture ofPart A and Part B can be sterilized by autoclaving.

In some embodiments, the soybean oil, which enhances clotting, isreplaced with chia bean oil which is anti-inflammatory and reducesclotting. In one embodiment, a pharmaceutical composition with soybeanoil is used in initial phase of the infusion in which bleeding isoccurring. A pharmaceutical composition with chia bean oil is used forlater stages of the infusion when bleeding is no longer an issue anissue.

In some other embodiments, the glycerol in Part A is replaced withmannitol. In other embodiments, the egg phospholipids is replaced withα-phosphatidylcholine to eliminate source of protein contamination andanaphylaxis due to contamination of egg phospholipid with egg protein.In yet other embodiments, the amino acids in Part B of Recipe 2 isreplaced with N-acetyl amino acids.

In one embodiment, the pharmaceutical composition is a non-oxygenatedpharmaceutical composition. As used herein, the term “non-oxygenatedpharmaceutical composition” refers to a formulation that is prepared inatmospheric air and is not loaded with oxygen by any oxygenation deviceor method.

In certain embodiments, the pharmaceutical composition may be loadedwith a lipophilic gas prior to clinical application. Examples of suchgases include, but are not limited to, oxygen, xenon, argon, nitricoxide, carbon monoxide, hydrogen sulfide. As used herein, “apharmaceutical composition loaded with a lipophilic gas” refers to apharmaceutical composition that has been subjected to a process toincrease the content of such lipophilic gas in the pharmaceuticalcomposition. A pharmaceutical composition may be loaded with alipophilic gas by bubbling the lipophilic gas through the pharmaceuticalcomposition for a desired period of time, or by agitating thepharmaceutical composition in the presence of the lipophilic gas underpressure.

In one embodiment, the pharmaceutical composition is oxygenated bybubbling pure oxygen or a gas with an oxygen content in the range of 21%to 100% (v/v), preferably 40% to 100% (v/v), more preferably 60% to 100%(v/v), and most preferably 80% to 100% (v/v), through the mixture for aperiod of 30 seconds or longer, preferably 1-15 minutes, more preferably1-5 minutes. Oxygen may also be added under pressure followed by areduction of the pressure to one atmosphere. In one embodiment, thepharmaceutical composition is oxygenated immediately prior toapplication. The pharmaceutical composition may be oxygenated usingportable oxygen tanks or portable oxygen concentrators, such the EvergoPortable Pulse Dose Oxygen concentrator produced by Philips Healthcareat Andover, Mass.

Another method could be allowing the emulsion to equilibrate with anatmosphere filled with the gas that is to be added. In most cases abubble trap would be necessary to remove bubbles that could become gasemboli. The equilibration time for a pharmaceutical composition of aparticular composition may be determined experimentally.

In one embodiment, the pharmaceutical composition comprises anoxygenated lipid emulsion. As used herein, the term “oxygenated lipidemulsion’ or “oxygenated pharmaceutical composition” refers to aspecific type of gassed lipid emulsion or gassed fluid which has beenforced to absorb oxygen such that the total concentration of oxygencontained therein is greater than that present in the same liquid atatmospheric equilibrium conditions.

Kits

Another aspect of the present invention relates to a resuscitation kit.In one embodiment, the resuscitation kit comprises an oxygenatedpharmaceutical composition and at least one additive. Examples ofadditives include, but are not limited to, oncotic agent, crystalloidagent, vessel expander, cardioplegic, or cardiotonic agent scavengers offree radicals or mediators, cell signaling modulators, and receptoragonists or antagonists. In another experiment, the kit further containsan intravenous infusion (IV) set. In another embodiment, the oxygenatedpharmaceutical composition is contained in one or more preloadedsyringes for emergency application.

In another embodiment, the kit contains a pharmaceutical composition anda portable oxygen container that can be used to re-oxygenate thepharmaceutical composition immediately prior to application. The oxygencontainer may contain pure oxygen, or a gas mixture of oxygen withhydrogen sulfide and/or carbon monoxide and/or nitric oxide. In anotherembodiment, the kit contains a pharmaceutical composition, and an airpump for oxygenating the pharmaceutical composition with ambient airimmediately prior to application.

In another embodiment, the kit contains pharmaceutical composition and aportable oxygen concentrator for oxygenating the pharmaceuticalcomposition with oxygen filtered from ambient air immediately prior toapplication. In one embodiment, the portable oxygen concentrator is anEvergo Portable Pulse Dose Oxygen concentrator produced by PhilipsHealthcare at Andover, Mass.

In another embodiment, the kit contains a pharmaceutical composition andan oxygen producing canister that is capable of producing oxygen througha chemical reaction. Chemicals that may be used for the production ofoxygen include, but are not limited to, sodium chlorate, sodium peroxideand potassium superoxide.

In another embodiment, the kit further contains a bubble removingdevice, such as a bubble trap.

In another embodiment, the kit contains syringes that are prefilled withthe pharmaceutical composition of the present invention and are ready tobe pushed into the blood stream of shock patients. In certainembodiment, the syringes have volumes of 60-500 cc. In otherembodiments, the syringes also come with delivery tubing. Thepharmaceutical composition may also be preloaded with oxygen, some othergas or other beneficial substances. In another embodiment, the kitcontains cartridges that are prefilled with the pharmaceuticalcomposition of the present invention. The cartridges can be snapped intoan apparatus that would infuse the pharmaceutical composition into apatient at a desired rate.

Treatment Methods

Another aspect of the present invention relates to a method for treatingconditions related to lack of blood supply with a lipid-basedpharmaceutical composition. Conditions related to a lack of blood supplyinclude, but are not limited to, hypovolemia, ischemia, hemodilution,trauma, septic shock, cancer, anemia, cardioplegia, hypoxia and organperfusion. The term “hypovolemia,” as used herein, refers to anabnormally decreased volume of circulating fluid (blood or plasma) inthe body. This condition may result from “hemorrhage,” or the escape ofblood from the vessels. The term “ischemia,” as used herein, refers to adeficiency of blood in a part of the body, usually caused by afunctional constriction or actual obstruction of a blood vessel.Conditions related to lack of blood supply also include situations inwhich the intravascular volume may be normal but blood vessels aredilated. Examples are septic shock in which mediators such asendothelium-derived relaxing factors cause blood vessel tone andresponsiveness to catecholamines to decrease. Examples of anendothelium-derived relaxing factor is nitric oxide. Prostaglandin E isanother molecule that dilates blood vessels.

The pharmaceutical composition may be administered intravenously,intra-arterially or intra cardiac to a subject in need of suchtreatment. Administration of the pharmaceutical composition can occurfor a period of seconds, hours, days or weeks depending on the purposeof the pharmaceutical composition usage, the ability to control bloodloss or the ability to restore spontaneous cardiac contraction. Forexample, when used as a blood volume expander and an oxygen carrier forthe treatment of severe hemorrhage shock, the usual time course ofadministration is as rapidly as possible, which may range from about 1ml/kg/hour to about 150 ml/kg/min or from about 10 ml/kg/min to about150 ml/kg/min. For the treatment of severe hemorrhagic shock, thepharmaceutical composition is given in an amount and at an infusion ratethat is sufficient to raise the blood pressure in the patient. Incertain embodiments, the pharmaceutical composition is given in anamount of 500-4000 ml, 500-2000 ml, 500-1000 ml, 1000-4000 ml and1000-2000 ml. In certain embodiments, the pharmaceutical composition isgiven in an amount that equals to about 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55% 60%, 65%, 70%, 75%, 80%, 85% or 90% of the normalblood volume of a patient in a period of 30-300 seconds. In certainembodiments, the pharmaceutical composition is given at a rate of200-4000 ml/min. In other embodiments, the pharmaceutical composition isgiven at a rate of about 1000-4000 ml/min. In other embodiments, thepharmaceutical composition is given at a rate of about 200-2000 ml/min.In other embodiments, the pharmaceutical composition is given at a rateof about 100-1000 ml/min. In yet other embodiments, the pharmaceuticalcomposition is given at a rate of about 500-700 ml/min. In someembodiments, the pharmaceutical composition is given withoutoxygenation. In other embodiments, the pharmaceutical composition is anoxygenated pharmaceutical composition. The infusion rate may vary from alow of 5 ml/min to 120 liters/hour. There is no upper limit of volume orrate due to the fact that blood loss can be massive and the infusion ofthe pharmaceutical composition would be given at a rate and volumenecessary to provide sufficient perfusion of tissues and tissueviability.

Another aspect of the present invention relates to a method forincreasing blood pressure in a subject in need of such treatment. Incertain embodiment, the blood pressure refers to systolic pressure. Themethod comprises administering into the patient an effective amount ofthe pharmaceutical composition of the present invention. In oneembodiment, the subject is a mammal who lost at least 15% of its bloodvolume. In another embodiment, the subject is a mammal who lost 15% to30% of its blood volume. In another embodiment, the subject is a mammalwho lost at least 30% of its blood volume. In another embodiment, thesubject is a mammal who lost 30% to 40% of its blood volume. In anotherembodiment, the subject is a mammal who lost at least 40% of its bloodvolume. In another embodiment, the subject is in hemorrhagic shock. Inyet another embodiment, the subject is in severe hemorrhagic shock. Asused herein, the term “hemorrhagic shock” refers to a shock statusinduced by the loss of at least 15% of the blood volume. As used herein,the term “severe hemorrhagic shock” refers to a shock status induced bythe loss of at least 30% of the blood volume.

In one embodiment, the “effective amount” of the pharmaceuticalcomposition needed to increase the blood pressure in a subject withhemorrhagic shock or severe hemorrhagic shock is 40%, 50%, 60%, 70%,80%, 90%, 100%, 120%, 150%, 200% or 300% of the lost blood volume.Typically, the pharmaceutical composition is infused initially at avolume that equals to at least 40%, 50%, 60%, 70% 80%, 90% or 100% ofthe lost blood volume to raise the blood pressure. A larger amount(e.g., 150% to 300% or even more) is given on an as needed basis.

In another embodiment, the “effective amount” of the pharmaceuticalcomposition needed to increase the blood pressure in a subject withhemorrhagic shock or severe hemorrhagic shock is the amount needed torestore the systolic pressure of the subject to 70 mmHg, 80 mmHg, 90mmHg, or higher.

Other hemodynamic parameters, such as perfusion of brain, kidneys,heart, muscle, spleen or other tissues, cardiac output, stroke volumeindex, mitochondrial oxidative phosphorylation followed by near infraredspectroscopy or other means, blood lactate or membrane polarization, mayalso be used to determine the “effective amount” of the pharmaceuticalcomposition needed to increase the blood pressure in a subject.

While the pharmaceutical composition of the present invention is beingadministered to and circulated through the subject, various agents suchas cardioplegic or cardiotonic agents may be administered eitherdirectly into the subject's circulatory system, administered directly tothe subject's myocardium, or added to the pharmaceutical composition thepresent invention. These components are added to achieve desiredphysiological effects such as maintaining regular cardiac contractileactivity, stopping cardiac fibrillation or completely inhibitingcontractile activity of the myocardium or heart muscle.

Cardioplegic agents are materials that cause myocardial contraction tocease and include anesthetics such as lidocaine, procaine and novocaineand monovalent cations such as potassium ion in concentrationssufficient to achieve myocardial contractile inhibition. Concentrationsof potassium ion sufficient to achieve this effect are generally inexcess of 15 mM.

During revival of a subject, the subject may be re-infused with amixture of the pharmaceutical composition described along with bloodretained from the subject or obtained from blood donors. Whole blood isinfused until the subject achieves an acceptable hematocrit, generallyequal to or exceeding hematocrits of about 30% and the goals ofresuscitation such as a normal lactate or mixed venous oxygen saturationof between 60% and 76% have been achieved. When these goals have beenachieved, rapid infusion is discontinued and the subject is revivedafter closure of surgical wounds using conventional procedures. Incertain embodiments, the pharmaceutical composition of the presentinvention is used to treat shock, including but are not limited to,neurogenic shock, cardiogenic shock, adrenal insufficiency shock andseptic shock.

Another aspect of this invention is ability to increase blood pressureby reversible absorption of nitric oxide. Hemoglobin and nitric oxidesynthase inhibitors are very effective at removing nitric oxide. Howeverthey do not readily lead to the release of nitric oxide. Such potent andirreversible uptake of nitric oxide can lead to lethal vasoconstrictioneven as the blood pressure increases. In contrast, the pharmaceuticalcomposition takes up nitric oxide because of the greater solubility ofnitric oxide in lipids. This mechanism is not as potent as theaforementioned methods and it is highly reversible. This mechanismmethod provides a variable reservoir of nitric oxide that may take up orrelease nitric oxide in a way that varies with local concentrations ofnitric oxide.

In certain other embodiments, the pharmaceutical composition isadministered. intraperitonealy, transdermally, enterally, rectally andby inhalation.

Another aspect of the present invention relates to a method of using thepharmaceutical composition described above to oxygenate patients whoselungs are severely damaged and unable to absorb oxygen even with specialmodes of ventilation. The oxygen loaded pharmaceutical composition maydeliver oxygen to tissues via circulation and allows the lung to recoverfrom the damage. In this regard, the pharmaceutical composition may beused to replace extracorporeal membrane oxygenation (ECMO).

Another aspect of the present invention relates to a method of using thepharmaceutical composition in exchange transfusion and whole circulationperfusion to wash out blood containing harmful materials such asinfectious agents, cancerous agents, and toxic agents. In both cases,the pharmaceutical composition may comprise an emulsion consisting of20% lipid micelles and isotonic saline with or without albumin. Thepharmaceutical composition may also be used to absorb toxic chemicalmolecules/biomolecules produced as the result of trauma, hemorrhagicshock or other form of shocks. Oxygen or other gases may or may not beadded.

Treatment of Seizure or Neuronal Injuries with Xenon/Argon-CarryingPharmaceutical Composition

Another aspect of the present invention relates to a method for treatingseizure or neuronal injury using xenon or argon carried by thepharmaceutical composition of the present invention. Xenon is colorless,heavy, odorless noble gas. Xenon has been used as a general anaesthetic.Two physiological mechanisms for xenon anesthesia have been proposed.The first one involves the inhibition of the calcium ATPase pump—themechanism cells use to remove calcium (Ca²⁺)—in the cell membrane ofsynapses (see e.g., Franks, J J et al. Anesthesiology 82:108-117). Thesecond mechanism involves the non-specific interactions between theanesthetic and the lipid membrane (see, e.g., Heimburg, T and Jackson AD, 2007, Biophysical Journal 92:3159-65. Xenon has a minimum alveolarconcentration (MAC) of 71%, making it 50% more potent than N₂O as ananesthetic. Thus it can be used in concentrations with oxygen that havea lower risk of hypoxia.

Xenon is also an antagonist of N-methyl-d-aspartate receptors (NMDAreceptors) and can be used to treat brain injuries. The NMDA receptorsexacerbate the damage from oxygen deprivation and xenon performs betteras a neuroprotectant than either ketamine or nitrous oxide, which haveundesired side-effects (Ma, D.; et al., 2002, British Journal ofAnaesthesia 89:739-746).

Without being bound by any theory, it is believed that glutamateexcitotoxicity is involved in the development of injury in conditionssuch as traumatic brain injury and ischemia/stroke and that xenonneuroprotection is mediated by competitive inhibition of the NMDAreceptor at its glycine-binding site.

Argon (Ar) is another noble gas that has shown neuroprotectiveproperties in in vitro models of cerebral ischemia and traumatic braininjury (Loetscher et al., Crit. Care 2009, 13:R206).

In one embodiment, a xenon- or argon-carrying pharmaceutical compositionis used to treat seizure, neuronal injuries and/or brain ischemia in asubject. The treatment comprises: infusing a sufficient amount of axenon- or argon-carrying pharmaceutical composition into said subjectfor a desired period of time. The xenon- or argon-carryingpharmaceutical composition may be produced by mixing degased lipidcomponent and degased polar liquid in a container filled with xenon, orby bubbling xenon through a degassed pharmaceutical composition. Asnoted earlier, the gas carrying capacity of the pharmaceuticalcomposition is proportional to its lipid content. Accordingly,pharmaceutical composition with high lipid content (e.g., 30-50% v/v) isused when a high Xenon dose is needed. In another embodiment, thepharmaceutical composition is saturated with a mixture of xenon andoxygen with the percentage loaded with xenon or argon from 10 to 80%(v/v). In one embodiment, the pharmaceutical composition is saturatedwith a mixture of xenon/argon and oxygen at a xenon/argon:oxygen ratioof 50:50, 60:40, 70:30, 75:25, or 80:20 (v/v). In yet anotherembodiment, the pharmaceutical composition is a mixture of a firstpharmaceutical composition saturated with xenon or argon and a secondpharmaceutical composition saturated with oxygen. In yet anotherembodiment, xenon or argon is used in conjunction with another volatileanesthetic, such as sevoflurane.

The treatable neuronal injuries involving NMDA receptors, include butare not limited to, ischemic stroke, intracranial bleeding due totrauma, or anoxic brain injury caused by blood loss or other forms ofshock or seizure disorder, and encephalitis.

In one embodiment, neuronal injuries are injuries mediated bymethyl-D-aspartate (NMDA)-type glutamate receptors. In anotherembodiment, the neuronal injuries are injuries caused by oxygendeprivation in the brain.

In certain embodiments the xenon, argon, Xe/O₂ or Ar/O₂-carryingpharmaceutical composition is infused at a rate of 20 to 100,000ml/hour, 500 to 100,000 ml/hour, or 5000 to 60,000 ml/hour. The infusionmay last for a period of several minutes to several weeks, depending onthe condition and specific needs of the patient. In certain embodiments,the xenon, argon, Xe/O₂ or Ar/O₂-carrying pharmaceutical composition isinfused for a period of 3 to 600 minutes, 5-300 minutes, 10-120 minutes,10 to 24 hours, or 1 to 7 days.

In another embodiment, the xenon-carrying pharmaceutical compositionalso carries a desired amount of carbon monoxide to prevent thedevelopment of pathologic conditions such as ischemia reperfusioninjury.

In another embodiment, the xenon-carrying pharmaceutical compositionalso carries a desired amount of hydrogen sulfide to regulate brainperfusion and promote organ survival.

In another embodiment, the xenon-carrying pharmaceutical composition isused in combination with hypothemia (32° C.-34° C.) for treatingneuronal injuries. Xenon may similarly be used in cardiac disease givenits utility in treating heart failure (Baumert J H, Falter F, Eletr D,Hecker K E, Reyle-Hahn M, Rossaint R. Xenon anaesthesia may preservecardiovascular function in patients with heart failure. ActaAnaesthesiol Scand. 2005 July; 49(6):743-9

In one embodiment, the xenon/argon treatment is given within 72 hours ofneuronal injury or brain ischemia.

In another embodiment, the xenon/argon treatment is given within 24hours of neuronal injury or brain ischemia.

In another embodiment, the xenon/argon treatment is given within 8 hoursof neuronal injury or brain ischemia.

In another embodiment, the xenon/argon treatment is given within 4 hoursof neuronal injury or brain ischemia.

In another embodiment, the xenon/argon treatment is given within 2 hoursof neuronal injury or brain ischemia.

Treatment of the Hypotension of Septic Shock with the PharmaceuticalComposition of the Present Invention

Another aspect of the present invention relates to a method for treatingseptic shock in a subject with pharmaceutical composition to absorbendogenously produced nitric oxide. Septic shock, or sepsis, is amedical condition in which acute inflammation, low blood pressure, andblood clotting cause a dangerous decrease in the delivery of blood tothe organs. Because of the failure of oxygen delivery to themicrociculation and the inability to utilize oxygen, once delivered totissues the patient's organs start to fail, one after the other.Currently, only supportive treatment is available. Nitric oxide plays aprominent role in the pathophysiology of both hemorrhagic and septicshock. Excess nitric oxide release from endothelial cells causerelaxation of arterial smooth muscle cells, loss of vascular tone andhypotension resulting in the inability to perfuse the microcirculation.In addition, nitric oxide can interact with reactive oxygen species andform toxic products such as peroxynitrite. In such cases it wouldbeneficial to remove excess nitric oxide. Therefore, in one embodiment,the method comprises administering into said subject, an effectiveamount of the pharmaceutical composition for a desired period of time toabsorb or remove endogenously released nitric oxide from the circulationsystem. The nitric oxide-absorbing pharmaceutical composition can beadministered intravenously or intra-arterially at a rate of about 20ml/hour to about 15,000 ml/min, preferably at a rate of 4000 ml/minute.

Treatment of Vasoconstriction and/or Hypertension with NitricOxide-Carrying Pharmaceutical Composition of the Present Invention

Some diseases such as Raynaud's disease occur because of increasedvascular resistance. Other examples are Prinzmetal's angina, cerebralvasospasm and atherosclerosis. In this case loading the pharmaceuticalcomposition with nitric oxide would relieve the vasoconstriction. Alsoin sickle cell crisis a combination of micelles loaded with nitric oxideand oxygen would be useful with the nitric oxide loaded micelles openingconstricted small vessels while oxygen loaded cells delivered oxygen toischemic tissues. Oxygen free radicals would be scavenged by theantioxidant moieties within the emulsion. In all embodiments thenitric-oxide carrying pharmaceutical composition also carries a desiredamount oxygen or other gases depending upon the physiological need. Inanother embodiment, the nitric-oxide carrying pharmaceutical compositionalso carries a desired amount of carbon monoxide to prevent thedevelopment of pathologic conditions such as ischemia reperfusioninjury.

In another embodiment, the nitric-oxide carrying pharmaceuticalcomposition also carries a desired amount of hydrogen sulfide to promoteperfusion of the brain and to modulate apoptosis.

Preservation of Organs

Another aspect of the present invention relates to a method ofpreserving the biological integrity of organs of a mammalian donororganism using the pharmaceutical composition described. In oneembodiment, the subject organ is chilled and the pharmaceuticalcomposition is perfused into the subject organ using a pumpedcirculating device such as a centrifugal pump, roller pump, peristalticpump or other known and available circulatory pump. The circulatingdevice is connected to the subject organ via cannulae insertedsurgically into appropriate veins and arteries. When the pharmaceuticalcomposition is administered to a chilled subject organ, it is generallyadministered via an arterial cannula and removed from the subject via avenous cannula and discarded or stored. In another embodiment, thepharmaceutical composition is perfused at a temperature in the roomtemperature range (15° C. to 25° C.). In another embodiment, thepharmaceutical composition is perfused at a temperature in the sub-bodytemperature range (25° C. to 34° C.). In another embodiment, thepharmaceutical composition is perfused at a temperature in the bodytemperature range (34° C. to 39° C.).

When used for organ perfusion during an organ transplantation, thepharmaceutical composition may be administered over a period of hours.In certain embodiments, the pharmaceutical composition comprises chiabean oil for its immunosuppressive effect.

Diagnosis

The pharmaceutical composition of the present invention may also be usedas a diagnostic tool. Because of its hydrophobic properties, thepharmaceutical composition is capable of absorbing lipophilic moietiesthat are produced in various disease states. Analysis of thepharmaceutical composition after it has circulated through the bloodstream can reveal the presence of diseases that release such moietiesinto the bloodstream. An example is the production of increased carbonmonoxide and nitric oxide in septic shock or other diseases that involvethe induction of heme oxygenase that leads to increased productionendogenous carbon monoxide.

EXAMPLES

The following example is put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tocarry out the method of the present invention and is not intended tolimit the scope of the invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperature,etc.), but some experimental error and deviation should be accountedfor. Unless indicated otherwise, parts are parts by weight, molecularweight is weight average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1 Methods and Materials

Lipid Emulsions Prepared by Sonication:

Lipid emulsion of various concentrations was prepared by mixing 1-3grams of soybean oil, 0.12 grams of soy lecithin, and 0.24 grams ofglycerol and water to make the volume up to 10 ml. This mixture was putinto a 15 ml vial and emulsified in an ice bath using an ultrasound celldisruptor.

Lipid Emulsions Prepared by Homogenization and Microfluidization:

An emulsion consisting of soybean oil (SIGMA, St. Louis) was prepared.The composition was composed of between 20%-50% w/v soybean oil,de-ionized water, varying amounts of an emulsifying agent (eitherphosphotidylcholine (Avanti Polar Lipids, Alabaster, Ala.) or egg yolkphospholipids (Sigma-Aldrich, St. Louis, Mo.), and with or without 5%human serum albumin (Sigma-Aldrich, St. Louis, Mo.). The albumin wasadded after the formation of the micelles. Some samples also contained154 mM NaCl. The mixture was homogenized for five minutes with aSilverson® high-shear rotor/stator homogenizer. A M-110T® microfluidizer(Microfluidics, Newton, Mass.) was used for final homogenization of thecoarse emulsion prepared by the Silverson homogenizer.

Commercially Obtained Lipid Emulsions:

20% Intralipid® (marketed and sold by Baxter International Inc.,Deerfield, Ill.) was used as a model lipid emulsion. It is composed of20% soy bean oil, 1.2% egg yolk phospholipids 2.25% glycerin, water andsodium hydroxide to adjust the pH to 8. Another model lipid emulsionobtained commercially is Liposyn® III 20% sold by Hospira in LakeForest, Ill. This emulsion consists of 20% soybean oil, 1.2% eggphospholipids, 2.5% glycerin, water and NaOH to adjust the pH to 8.3.

Determination of Oxygen Content of Emulsions Using Mass Spectroscopy:

The technique developed in Dr. Juan Rodriguez' laboratory was employed(Cornelius J, Tran T, Turner N, Piazza A, Mills L, Slack R, Hauser S,Alexander J S, Grisham M B, Feelisch M, Rodriguez J. “Isotope tracingenhancement of chemiluminescence assays for nitric oxide research” Biol.Chem. February; 390(2):181-9 (2009)). Some samples (1 ml each) were leftopen to air in 2.0 ml tubes for 30 minutes prior to dissolved gasanalysis. In other experiments the emulsion was placed into a 15 mltube. The headspace was filled with the gas under study and the gas wasallowed to enter the emulsion by equilibration over 60 minutes with thetube on its side to increase the gas emulsion interaction. 50 μL drawnfrom each of these fluids were injected into a Sievers purge vessel at37° C. containing 36 ml of a mildly acidic solution consisting of 32 mlof 1M HCL and 4 ml of 0.5M ascorbic acid. The solution was continuouslypurged with high purity helium to transport any oxygen released from thesamples to a mass spectrometer (HP 5975) for direct gas analysis.Signals generated at m/z=32 upon injection of RL and lipid emulsionsamples were integrated using Peakfit and compared to those obtainedwith distilled water.

Animals and Animal Procedures:

Male and female mice weighing 27-47 grams were utilized. The strainswere either CD-1 or NFR2. All comparisons utilized the same strain. Micewere anesthetized using ketamine/xylazine anesthesia administeredsubcutaneously. In order to prevent the skewing of data due to thecardiodepressant effects of the anesthetic agent, the experiment wasaborted and the mouse euthanized in the rare instance when moreanesthetic was required than the calculated dose. Once it was clear thatthe mouse was well-anesthetized, the carotid artery was cannulated. Asmuch blood as possible was removed in three minutes. This resulted inthe loss of 55% of blood volume and 100% lethality without any infusion.Immediately after blood removal infusions were administered over twominutes.

Pharmaceutical composition, resuscitation fluid (Ringer's lactate) orshed blood was administered at a volume equal to the amount of bloodthat had been removed. Blood pressure was measured at the carotid arteryusing a BP-2 monitor made by Columbus Instruments (Columbus, Ohio). Thismonitor measures the blood pressure as a voltage. A standard curve wasprepared. Measured voltages were converted to blood pressure (BP) usingthe following formula:BP=[Voltage−0.1006]/0.0107

No warming measures were applied to the mice. No measures were taken tosupport respiration.

Rat Model of Hemorrhagic Shock

Experiments using rats were approved by the University of TennesseeHealth Science Center Animal Care and Use Committee. Sprague-Dawley ratsweighing between 200 and 300 g were purchased with the carotid arteryand ipsilateral jugular vein pre-cannulated. The rats were givenketamine/xylazine anesthesia subcutaneously. After the effectiveness ofthe anesthetic was assured the carotid cannula was connected with ablood pressure monitor. The rats were bled to a mean blood pressure of40 mmHg and maintained at that level for 45 minutes by the removal orinfusion of shed blood as needed. At the end of the 45 minute period ofshock the test pharmaceutical composition was infused in a volume equalto the amount of blood removed. The blood pressure was recorded for 45minutes after resuscitation after which surviving rats were euthanizedby exsanguination.

Statistical Analysis:

Data were analyzed using the two-tailed, Student's unpaired t test.

Example 2 Oxygen and Nitric Oxide Content of the PharmaceuticalComposition of the Present Invention

Intralipid® 20% I.V. Fat Emulsion (marketed and sold by BaxterInternational Inc., Deerfield, Ill.) was used as a sample pharmaceuticalcomposition (LM). The composition of Intralipid® is 20% soybean oil,1.2% egg yolk phospholipids, 2.25% glycerin, water and sodium hydroxideto adjust the pH to 8. Oxygen content in the samples was measured usingmass spectrometry. As shown in Table I, the oxygen content of LM wasnearly twice that of Ringer's lactate (RL), a standard resuscitationfluid infused when a large amount of blood is lost. The oxygen contentof water was assigned the value of 1. The oxygen content of RL wasslightly less than that of water. As shown in Table 2, the oxygencontent of the LM was increased five-fold by bubbling oxygen through itfor approximately 1 minute. After oxygen loading, the oxygen content ofLM compared favorably to that of blood with the minimum acceptablehemoglobin level (i.e., 7.0 g/dl).

TABLE 1 Oxygen content of Ringer's lactate and Intralipid ® 20%. Eachmean is the result of 3 samples. Ringer's lactate Intralipid ® 20%Oxygen Content* 0.91 ± 0.11* 1.78 ± 0.09* *the oxygen content isexpressed as the amount relative to the oxygen content in water.

TABLE 2 Oxygen solubility in various liquids at 1 atm Oxygen Content at25° C. and Sea level Pressure Blood (hemoglobin of 7.0) 72.8 mg/L Water8.3 mg/L⁺ LM (20%) 15.1 mg/L LM (20% after oxygen perfusion) 75.5 mg/L⁺Obtained from “The Engineering Toolbox” Oxygen Solubility in Fresh andSea water athttp://www.engineeringtoolbox.com/oxygen-solubility-water-d_841.html.

These data show that LM may be loaded with sufficient oxygen to providefor aerobic metabolism. This point is further underscored by the factthat only 30% of the oxygen bound to hemoglobin is used under normalcircumstances. In extreme stress such as severe hemorrhagic shock 60%might be offloaded from hemoglobin. In contrast all of the oxygendissolved in LM should be available given the fact that oxygen isreleased 3 times faster from LM than from hemoglobin. The publishedrelease time for hemoglobin is 17.5 msec while the off time measuredwhen we purged oxygen from LM was less than 5 msec.

FIG. 2 shows the linear dependence of emulsion oxygen content on theconcentration of lipid micelles. Micelles were prepared by sonicating amixture of 1-3 grams of soybean oil, 0.12 gram of soybean lecithin and0.12 gram of glycerol and water to make a volume of 10 ml. The mixturewas put into a 15 ml tube and emulsified in an ice bath using anultrasound disruptor. Each point is the mean of 2-3 experiments. The Yaxis is the oxygen content of the emulsion relative to water. The X axisis the soybean oil concentration of the emulsion.

Example 3 Nitric Oxide Uptake of the Pharmaceutical Composition of thePresent Invention

In another set of experiments we measured the uptake of nitric oxide bya commercially available 20% soybean oil emulsion, Liposyn III 20%. Thisemulsion contains 20% soybean oil, 1.2% egg phospholipids and 2.5%glycerin in water. Using NaOH pH was adjusted to 8.3. Oxygen was addedby equilibration in which 200 μl of the emulsion was placed into a 15 mlvial. 100 ppm nitric oxide in helium was used to purge the headspace fortwo minutes taking care not to bubble the fluid. For sixty minutes thesample was allowed to reach gas-liquid equilibrium with the tube placedon its side to maximize gas-emulsion contact. The results are shown intable 3 below.

TABLE 3 NO Content of a 20% Soybean Oil Emulsion (Liposyn III 20%) andWater in moles/liter Emulsion Water 3.03 1.52 2.42 1.63 3.81 1.72 3.301.69 2.50 2.29 Mean 3.01 1.77 S.E 0.26 0.14

Example 4 The Effect of the Pharmaceutical Composition of the PresentInvention in Restoring Arterial Pressure in Mice with Severe HemorrhagicShock

The effect of the pharmaceutical composition (LM) in Example 2 on bloodpressure was determined in mice. Mice were anesthetized and a cannulawas placed into the carotid artery. All the blood that could be removedwas removed via the carotid artery. After the blood was removed a volumeof either RL or LM was given equal to the amount of blood removed. 6mice were in the LM group and 6 mice were in the RL group. Theobservation period was one hour. Two of the mice given RL died withinten minutes. All mice given LM lived through the entire hour observationperiod and until euthanized at 1-4 hours. Animals were euthanizedwhenever they began to awaken from the anesthesia or at the end of theobservation period to prevent suffering.

FIGS. 3 and 4 show the difference between the systolic blood pressure(FIG. 3) and diastolic blood pressure (FIG. 4) after hemorrhage andafter infusion of RL or LM at time=0, 30 and 60 minutes. The Y axisrepresents the blood pressure attained after infusion minus the bloodpressure after hemorrhage in mm of Hg. The X axis shows the specifictime after the infusion. All data were analyzed for statisticalsignificance using an unpaired two tailed t test. These graphs show thatLM raised the blood pressure higher than RL (p<0.01).

In another experiment, a pharmaceutical composition containingIntralipid® 20% and 5% (w/v) albumin was prepared by dissolving albumin(Sigma Aldrich, 99% pure, fatty acid free, essentially globulin free,catalog number A3782-5G) in Intralipid® 20% to a final concentration of50 mg/ml. The new pharmaceutical composition with albumin (VS) wastested using the experimental procedure described above. Albumindissolved in normal saline (NSA) and Ringer's lactate (RLA) at 50 mg/ml,as well as the shed blood (i.e., the blood that had been removed fromthe mice), were used as controls. In FIG. 5, the Y axis shows the meanblood pressure (expressed as percentage of mean pre-hemorrhage bloodpressure) achieved by infusion of the various fluids. The X axis showsspecific times after the infusion. The data show that VS is superioreven to shed blood in maintaining blood pressure. Similar results werealso obtained for the diastolic blood pressure (not shown). For eachtime point, an average of 6-7 mice is plotted. Differences between shedblood and VS was statistically significant (P<0.05) at 5, 15 and 30minutes.

Example 5 The Effect of the Pharmaceutical Composition of the PresentInvention in Restoring Arterial Pressure in Rats with Severe HemorrhagicShock

In another experiment anesthetized rats were bled to a mean pressure of40 that was maintained for 45 minutes using shed blood. At the end ofthis period a test fluid was given within 2 minutes. The test fluidswere the (1) emulsion containing 20% Intralipid mixed with 5% albuminNaCl 102 mM, Na(L) lactate 28 mM, KCL 4 mM, (2) Ringer's lactate, (3)Hextend and (4) shed blood. The results are shown in Table 4.

TABLE 4 Effect of the pharmaceutical composition in restoring arterialpressure in rats with severe hemorrhagic shock Ringer's Emulsion HextendLactate Blood Number of rats 6 2 2 2 Avg survival time (min) 35.8 +/−6.7 10.74 18.25 35.75 Avg mean arterial pressure 64.2 +/− 2.4 38.3 34.264.7

Hematoxylyn and eosin histopathological examination of the lung, liver,kidneys, spleen heart and intestine was carried out. No harmful effectof the emulsion was found.

These experimental results are consistent with the fact that the lipidmicelles in the pharmaceutical composition are capable of exerting anosmotic force and absorbing mediators of vascular patency, such asprostaglandins, nitric oxide, leukotrienes, and hromboxane.

Example 6 The Effect of Amino Acid-Containing Pharmaceutical Compositionin Restoring Arterial Pressure in Rats with Severe Hemorrhagic Shock

In another experiment, hemorrhagic shock was induced in mice asdescribed in Example 4. The mice were then resuscitated with a controlemulsion containing 20% Intralipid, 102 mM NaCl, 28 mM Na(L) lactate, 4mM KCL, or an test emulsion containing the control emulsion and 1 mMhistidine or tyrosine or phenylalanine or cysteine. Table 4 shows theeffect of the pharmaceutical composition in restoring arterial pressurein mice with hemorrhagic shock.

TABLE 5 Effect of the pharmaceutical composition in restoring arterialpressure in mice with hemorrhagic shock. Post infusion - # pressure inmice Pre bleed Shock shock IL alone 4 105.0 +/− 3.6  22.8 +/− 2.8 32.3+/− 6.8 79.0 +/− 2.3 14.5 +/− 2.4  8.3 +/− 3.1 IL + 2 112.0 +/− 13.016.04 +/− 0   63.0 +/− 4.0 Histidine 83.0 +/− 7.0 10.5 +/− 0.5 43.0 +/−0  IL + 2  134 +/− 18.0 24.0 +/− 1.0 68.0 +/− 3.0 Tyrosine 83.0 +/− 16 15.0 +/− 1.0 56.5 +/− 2.5 IL + 2 106.5 +/− 21.5 19.5 +/− 2.5 55.5 +/−2.5 Phenylalanine  70.5 +/− 14.5 13.5 +/− 0.5 42.0 +/− 6.0 IL + 2 156.5+/− 37.5 25.0 +/− 0  58.5 +/− 3.5 cysteine  99.0 +/− 14.0 16.0 +/− 3.045.0 +/− 5.0

The terms and descriptions used herein are set forth by way ofillustration only and are not meant as limitations. Those skilled in theart will recognize that many variations are possible within the spiritand scope of the invention as defined in the following claims, and theirequivalents, in which all terms are to be understood in their broadestpossible sense unless otherwise indicated.

What is claimed is:
 1. A pharmaceutical composition comprising: a lipidcomponent; an amphiphilic emulsifier; and a polar liquid carrier,wherein the lipid component and the amphiphilic emulsifier formfree-moving lipid-carrying micelles (LMs) or liposomes in the polarliquid carrier, wherein the pharmaceutical composition is free ofhemoglobin and fluorocarbon, and wherein the pharmaceutical compositionfurther comprises an effective amount of oxygen for enhancement ofaerobic metabolism.
 2. The pharmaceutical composition of claim 1,wherein said LMs or liposomes have an average diameter of 300 nm orsmaller.
 3. The pharmaceutical composition of claim 1, wherein said LMsor liposomes have an average diameter of 100 nm or smaller.
 4. Thepharmaceutical composition of claim 1, wherein said LMs or liposomeshave an average diameter of 30 nm or smaller.
 5. The pharmaceuticalcomposition of claim 1, further comprising an amino acid at a finalconcentration of 0.2-50 mM.
 6. The pharmaceutical composition of claim1, further comprising glycerol or mannitol in an amount of 2-5% (w/v).7. The pharmaceutical composition of claim 1, further comprisingglycerol or mannitol in an amount of about 2.25% (w/v).
 8. Thepharmaceutical composition of claim 1, further comprising albumin in anamount of 3-7% (w/v).
 9. The pharmaceutical composition of claim 1,further comprising albumin in an amount of about 5% (w/v).
 10. Thepharmaceutical composition of claim 1, wherein said amphiphilicemulsifier is egg yolk phospholipids or α-phosphatidylcholine in anamount of 1%-5% (w/v).
 11. The pharmaceutical composition of claim 10,wherein said amphiphilic emulsifier is egg yolk phospholipids orα-phosphatidylcholine in an amount of about 2% (w/v).
 12. Thepharmaceutical composition of claim 1, wherein said lipid component isselected from the group consisting of soybean oil, chia bean oil,pumpkin oil, flaxseed oil, fish oil and algae oil in an amount of10%-70% (w/v) of said pharmaceutical composition.
 13. The pharmaceuticalcomposition of claim 12, wherein said lipid component is selected fromthe group consisting of soybean oil, chia bean oil and algae oil in anamount of about 35% (w/v) of said pharmaceutical composition.
 14. Thepharmaceutical composition of claim 1, wherein said composition is freeof calcium, magnesium and potassium.
 15. The pharmaceutical compositionof claim 1, wherein said composition is free of aluminium.
 16. Thepharmaceutical composition of claim 1, wherein said LMs or liposomeshave an average diameter of 2-300 nm.
 17. The pharmaceutical compositionof claim 1, further comprising an amino acid at a final concentration of0.1 fM-200 mM.
 18. The pharmaceutical composition of claim 1, furthercomprising an amino acid at a final concentration of 0.1 fM-10 mM. 19.The pharmaceutical composition of claim 1, further comprising an aminoacid at a final concentration of 0.01 mM-200 mM.
 20. The pharmaceuticalcomposition of claim 1, further comprising an amino acid at a finalconcentration of 10 nM-10 μM.
 21. The pharmaceutical composition ofclaim 1, further comprising effective amounts of oxygen and nitric oxidefor regulation of vascular function and cellular metabolism.
 22. Thepharmaceutical composition of claim 1, further comprising erythrocyteghosts that traps said lipid component.
 23. The pharmaceuticalcomposition of claim 1, further comprising β-endorphin at a finalconcentration of 0.01-100 nM.
 24. The pharmaceutical composition ofclaim 1, further comprising 0.1 fM-10 mM histidine, cysteine, or anoligopeptide containing histidine or glycylglycine.
 25. Thepharmaceutical composition of claim 1, further comprising 0.01-0.2 Mhistidine.
 26. The pharmaceutical composition of claim 1, wherein saidlipid carrying micelles or liposomes have an average diameter of 90-120nm.
 27. The pharmaceutical composition of claim 1, wherein said lipidcarrying micelles or liposomes have an average diameter of 90-100 nm.28. The pharmaceutical composition of claim 1, wherein said lipidcarrying micelles or liposomes have an average diameter of 100-110 nm.29. The pharmaceutical composition of claim 1, wherein said lipidcarrying micelles or liposomes have an average diameter of 110-120 nm.30. The pharmaceutical composition of claim 1, further comprising one ormore oncotic agents.
 31. The pharmaceutical composition of claim 1,further comprising one or more immunomodulatory agents.
 32. Thepharmaceutical composition of claim 1, further comprising one or moreanti-inflammatory agents.
 33. The pharmaceutical composition of claim 1,further comprising one or more coagulation enhancers.
 34. Thepharmaceutical composition of claim 1, further comprising one or moreadditives selected from the group consisting of antioxidants,antibiotics, anti-fungal agents, vitamins, amino acids, vesselexpanders, surfactants, antibodies and mediators of vascular potency.